US20080006770A1 - Mass spectrometric imaging method under ambient conditions using electrospray-assisted laser desorption ionization mass spectrometry - Google Patents
Mass spectrometric imaging method under ambient conditions using electrospray-assisted laser desorption ionization mass spectrometry Download PDFInfo
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- US20080006770A1 US20080006770A1 US11/780,055 US78005507A US2008006770A1 US 20080006770 A1 US20080006770 A1 US 20080006770A1 US 78005507 A US78005507 A US 78005507A US 2008006770 A1 US2008006770 A1 US 2008006770A1
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/0004—Imaging particle spectrometry
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0459—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for solid samples
- H01J49/0463—Desorption by laser or particle beam, followed by ionisation as a separate step
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/10—Ion sources; Ion guns
- H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
- H01J49/165—Electrospray ionisation
Definitions
- the invention relates to molecular imaging, more particularly to mass spectrometric imaging under ambient conditions using electrospray-assisted laser desorption ionization mass spectrometry.
- Imaging mass spectrometry is widely used in the investigation of chemical or molecular distributions of solid samples, such as metals, polymers, semiconductors, and geological substances. Many attempts have been made to explore the feasibility of using imaging mass spectrometry in studying spatial distribution of proteins in various organs. However, due to the biological nature of target protein, e.g., being more labile to ionization energy and being in a state of flux, such efforts did not prove to be satisfactory.
- SIMS secondary ion mass spectrometry
- MALDI-MS matrix-assisted laser desorption ionization mass spectrometry
- DESI-MS desorption electrospray ionization mass spectrometry
- the object of the present invention is to provide a mass spectrometric imaging method that can be conducted under ambient conditions, and that can be used to obtain an imaging profile of a sample that has a self-sustained shape with speed and accuracy.
- a further object of the present invention is to provide a mass spectrometric imaging method that is capable of swiftly and un-obstructively maneuvering a sample to move relative to a desorption mechanism such that mass spectrometric results for substantially continuous areas of the sample can be obtained in a desirable short period of time.
- Another object of the present invention is to provide a mass spectrometer for implementing the mass spectrometric imaging method.
- a mass spectrometric imaging method includes the steps of: forcing sequentially generated charge-laden liquid drops to move towards a receiving unit of a mass spectrometer along a traveling path; scanning a sample with a laser beam which has an irradiation energy sufficient to cause analytes contained in the sample to be desorbed to fly along a plurality of flying paths respectively; and positioning the sample relative to the laser beam to render the plurality of flying paths intersecting the traveling path so as to permit a plurality of the analytes respectively along the plurality of flying paths to be occluded in a plurality of the charge-laden liquid drops respectively to thereby form a plurality of corresponding ionized analytes.
- the mass spectrometric imaging method further includes the steps of obtaining a plurality of mass spectra respectively for a plurality of scanned areas of the sample through analyzing the plurality of corresponding ionized analytes which respectively correspond to the plurality of scanned areas of the sample; selecting at least one representative mass-to-charge ratio (m/z) signal which may signify a characteristic of the sample from the plurality of mass spectra; and constructing an imaging profile for the sample based on intensities at each of the at least one representative mass-to-charge ratio signal displayed by the plurality of scanned areas.
- m/z mass-to-charge ratio
- a mass spectrometric system which is capable of obtaining an imaging profile, and which includes a mass spectrometer for analyzing ionized analytes.
- the mass spectrometric system includes: a receiving unit for the mass spectrometer; means for forcing sequentially generated charge-laden liquid drops to move towards the receiving unit along a traveling path; means for scanning a sample with a laser beam which has an irradiation energy sufficient to cause analytes contained in the sample to be desorbed to fly along a plurality of flying paths respectively; and means for positioning the sample relative to the laser beam to render the plurality of flying paths intersecting the traveling path so as to permit a plurality of the analytes respectively along the plurality of flying paths to be occluded in a plurality of the charge-laden liquid drops respectively to thereby form a plurality of corresponding ionized analytes
- FIG. 1 is a schematic diagram of a mass spectrometric system for implementing the preferred embodiment of a mass spectrometric imaging method under ambient conditions using electrospray-assisted laser desorption ionization mass spectrometry (ELDI-MS) according to the present invention
- ELDI-MS electrospray-assisted laser desorption ionization mass spectrometry
- FIG. 2 is a fragmentary schematic view of the mass spectrometric system
- FIG. 3 is a flow chart of the preferred embodiment of the mass spectrometric imaging method
- FIG. 4 ( a ) illustrates a photograph of a glossy ganoderma slice obtained for conducting imaging mass spectrometric analysis in exemplary example 1;
- FIGS. 4 ( b ) ⁇ ( h ) illustrate molecular imaging profiles constructed for the glossy ganoderma slice in exemplary example 1 ;
- FIG. 5 ( a ) ⁇ ( h ) illustrate negative images of FIGS. 4 ( a ) ⁇ ( h ), respectively;
- FIGS. 6 ( a ) ⁇ ( d ) illustrate four mass spectra of the glossy ganoderma slice obtained at various scanned areas thereof in exemplary example 1;
- FIG. 7 ( a ) is a photograph of an antrodia camphorata slice obtained for conducting imaging mass spectrometric analysis in exemplary example 2;
- FIGS. 7 ( b ) ⁇ ( d ) illustrate three mass spectra of the antrodia camphorata slice obtained at various scanned areas thereof in exemplary example 2;
- FIG. 8 ( a ) is another photograph of the antrodia camphorata slice
- FIGS. 8 ( b ) ⁇ ( x ) illustrate molecular imaging profiles constructed for the antrodia camphorata slice in exemplary example 2;
- FIG. 9 illustrates a mass spectrum obtained for one of two angelica sinensis diels slices, which were obtained for conducting imaging mass spectrometric analysis in exemplary example 3;
- FIG. 10 ( a ) illustrates a photograph of the angelica sinensis diels slices
- FIGS. 10 ( b ) ⁇ ( n ) illustrate molecular imaging profiles constructed for the angelica sinensis diels slices in exemplary example 3;
- FIGS. 11 ( a ) ⁇ ( n ) illustrate negative images of FIGS. 10 ( a ) ⁇ ( n ), respectively;
- FIG. 12 ( a ) is a diagram of a chicken brain slice obtained for conducting imaging mass spectrometric analysis in exemplary example 4.
- FIGS. 12 ( b ) ⁇ ( e ) illustrate four mass spectra of the chicken brain slice obtained at various scanned areas thereof in exemplary example 4;
- FIG. 13 ( a ) illustrate a diagram of the chicken brain slice with an Optical Cutting Temperature (OCT) drug that surround the periphery of the chicken brain slice;
- OCT Optical Cutting Temperature
- FIGS. 13 ( b ) ⁇ ( h ) illustrate molecular imaging profiles constructed for the chicken brain slice in exemplary example 4;
- FIGS. 14 ( a ) ⁇ ( h ) illustrate negative images of FIGS. 13 ( a ) ⁇ ( h ), respectively,
- FIG. 15 ( a ) illustrate a mass spectrum of a chicken heart slice, which is obtained for conducting imaging mass spectrometric analysis in exemplary example 5, at a location corresponding to fat surrounding an outer periphery of the chicken heart slice;
- FIG. 15 ( b ) illustrate a mass spectrum of the chicken heart slice at a location corresponding to muscle tissues at inner portions of the chicken heart slice
- FIG. 16 ( a ) illustrate a photograph of the chicken heart slice
- FIGS. 16 ( b ) ⁇ ( m ) illustrate molecular imaging profiles constructed for the chicken heart slice in exemplary example 5;
- FIGS. 17 ( a ) ⁇ ( m ) illustrate negative images of FIGS. 16 ( a ) ⁇ ( m ), respectively.
- a mass spectrometric system is used to implement the preferred embodiment of a mass spectrometric imaging method under ambient conditions using electrospray-assisted laser desorption ionization mass spectrometry (ELDI-MS) according to the present invention.
- the mass spectrometric system includes an electrospray unit 2 , a laser desorption unit 3 , a sample stage unit 4 , a receiving unit 5 , and an imaging processing software (not shown).
- the electrospray unit 2 includes a reservoir 21 for accommodating a liquid electrospray medium, and a nozzle 22 which is disposed downstream of the reservoir 21 , and which is configured to sequentially form liquid drops of the electrospray medium thereat for traveling along a traveling path.
- the electrospray unit 2 further includes a pump 23 disposed downstream of the reservoir 21 and upstream of the nozzle 22 for drawing the electrospray medium into the nozzle 22 .
- the nozzle 21 is spaced apart from the receiving unit 5 in a longitudinal direction (X) so as to define the traveling path.
- the electrospray unit 2 further includes a voltage supplying member 24 that is disposed to establish between the nozzle 22 and the receiving unit 5 a potential difference which is of an intensity such that the sequentially formed liquid drops are laden with a plurality of charges, and such that the charge-laden liquid drops are forced to leave the nozzle 22 for heading toward the receiving unit 5 along the traveling path.
- the electrospray medium is an acidified methanol solution (50%).
- the charges laden in the liquid drops can be either univalent or multivalent.
- the laser desorption unit 3 includes a laser transmission mechanism 31 that is capable of transmitting a laser beam 34 , a lens 32 that is disposed to receive the laser beam 34 from the laser transmission mechanism 31 for focusing the energy carried by the laser beam 34 , and a reflector 33 that is disposed to change the path of the laser beam 34 .
- the laser desorption unit 3 is adapted to irradiate a sample 6 such that, upon irradiation, a plurality of analytes, such as chemical or biochemical molecules, contained in the sample 6 are desorbed to fly along a plurality of flying paths, respectively.
- the laser transmission mechanism 31 is a nitrogen (N 2 ) gas laser (337 nm, 100 ⁇ J, Q-switch).
- the sample stage unit 4 includes a movable sample stage 41 and a computer-controlled positioning mechanism 42 .
- the sample stage 41 is movable relative to the laser beam 34 such that a laser spot may be formed at a different location on the sample 6 for each laser pulse.
- the computer-controlled positioning mechanism 42 is connected electrically to the sample stage 41 for controlling movement of the sample stage 41 relative to the laser beam 34 .
- the sample stage 41 is movable in a plane along the longitudinal direction (X) and a transverse direction (Y) perpendicular to the longitudinal direction (X). It should be noted herein that the sample stage 41 can be movable in three dimensions in other embodiments of the present invention.
- sample stage 41 can also be made stationary, while the laser beam 34 irradiated by the laser desorption unit 3 is made movable, in other embodiments of the present invention, as long as relative movement between the sample stage 41 and the laser beam 34 can be established.
- the sample stage 41 along with the sample 6 placed thereon, is positioned relative to the laser beam 34 to render the plurality of flying paths of the analytes to intersect the traveling path of the charge-laden liquid drops so as to permit a plurality of the analytes respectively along the plurality of flying paths to be occluded in a plurality of the charge-laden liquid drops respectively to thereby form a plurality of corresponding ionized analytes.
- the ionized analytes are formed due to passing of the charges in the liquid drops onto the analytes as the charge-laden liquid drops dwindle in size when approaching the receiving unit 5 along the traveling path.
- the receiving unit 5 includes a mass analyzer 51 formed with a conduit 52 that is in air communication with the environment, and a detector 53 for receiving signals generated by the mass analyzer 51 .
- the mass analyzer 51 receives the ionized analytes through the conduit 52 , separates the ionized analytes according to their m/z values (mass-to-charge ratios), and generates corresponding signals for the ionized analytes.
- the mass analyzer 51 is selected from the group consisting of an ion trap mass analyzer, a quadrupole time-of-flight mass analyzer, a triple quadrupole mass analyzer, an ion trap time-of-flight mass analyzer, a time-of-flight/time-of-flight mass analyzer, and a Fourier transform ion cyclotron resonance (FTICR) mass analyzer.
- an ion trap mass analyzer a quadrupole time-of-flight mass analyzer, a triple quadrupole mass analyzer, an ion trap time-of-flight mass analyzer, a time-of-flight/time-of-flight mass analyzer, and a Fourier transform ion cyclotron resonance (FTICR) mass analyzer.
- FTICR Fourier transform ion cyclotron resonance
- the preferred embodiment of the mass spectrometric imaging method according to the present invention is performed in conjunction with the mass spectrometric system described above.
- step 11 an object of self-sustained shape is first cut into a thin slice, which is referred to as the sample 6 , by a sharp razor blade or through the method of frozen section, and the sample 6 is then placed on a stainless steel sample plate 7 , which is disposed on the sample stage 41 of the sample stage unit 4 .
- step 12 sequentially generated charge-laden liquid drops are forced to move towards the receiving unit 5 along a traveling path.
- the sequentially generated charge-laden liquid drops are formed by the electrospray unit 2 at the nozzle 1 thereof, and are forced to move towards the receiving unit 5 by the electrospray unit 2 .
- the sample 6 is irradiated with the laser beam 34 which has an irradiation energy sufficient to cause analytes contained in the sample 6 and located at the laser spot to be desorbed to fly along a corresponding flying path.
- the laser beam 34 is transmitted through a fiber optic unit.
- step 14 the sample 6 is positioned relative to the laser beam 34 to render the flying path to intersect the traveling path so as to permit the desorbed analytes along the flying path to be occluded in the charge-laden liquid drops to thereby form a plurality of corresponding ionized analytes.
- step 15 a mass spectrum is obtained for the scanned area of the sample 6 (i.e., the area where the laser spot is formed) through analyzing the corresponding ionized analytes which correspond to the scanned area of the sample 6 .
- step 16 the sample 6 is moved relative to the laser beam 34 such that a different area of the sample 6 is irradiated by the laser beam 34 .
- Steps 14 to 16 are then repeated multiple times in a traceable manner.
- various areas of the sample 6 are irradiated by the laser beam 34 to generate corresponding ionized analytes, and to obtain corresponding mass spectra.
- the laser beam 34 is kept to irradiate along a predetermined line, and the sample stage 41 of the sample stage unit 4 is moved relative to the laser beam 34 in incremental steps in the longitudinal direction (X) and the transverse direction (Y) by control of the computer-controlled positioning mechanism 42 , so as to position the sample 6 relative to the laser beam 34 .
- the computer-controlled positioning mechanism 42 controls the sample stage 41 to move in incremental steps in the transverse direction (Y), such that various areas of the sample 6 , which is placed on the sample stage 41 , are sequentially irradiated by the laser beam 34 .
- analytes contained in the sample 6 at the scanned area are desorbed to fly along the corresponding flying path that is rendered to intersect the traveling path of the charge-laden liquid drops so as to form the corresponding ionized analytes, and a corresponding mass spectrum is then obtained through analyzing the corresponding ionized analytes which correspond to the scanned area of the sample 6 .
- the laser beam 34 forms a laser spot with an area of 100 ⁇ m ⁇ 150 ⁇ m on the sample 6
- the laser transmission mechanism 31 has an operating frequency of between 5 Hz to 10 Hz.
- step 17 at least one representative mass-to-charge ratio (m/z) signal which may signify a characteristic of the sample 6 is selected from the plurality of mass spectra.
- a molecular imaging profile for the sample 6 is constructed based on intensities at each of the at least one representative m/z signal displayed by the plurality of scanned areas of the sample 6 .
- the present invention is described hereinafter in conjunction with a number of exemplary examples conducted to verify the mass spectrometric imaging under ambient conditions using electrospray-assisted laser desorption ionization mass spectrometry. It should be noted herein that the exemplary examples are for illustrative purposes only, and should not be taken as limitations imposed on the present invention.
- FIG. 4 ( a ) and FIG. 5 ( a ) a slice of glossy ganoderma, a genus of polypores, was obtained for exemplary example 1 using a razor blade, where FIG. 4 ( a ) shows a photograph of the glossy ganoderma slice and FIG. 5 ( a ) is a negative image of FIG. 4 ( a ).
- the glossy ganoderma slice was measured 10 mm in length, 35 mm in width, and 3 mm in thickness.
- the glossy ganoderma slice was placed on the sample stage 41 of the sample stage unit 4 (refer to FIG. 1 ) to be irradiated by the laser beam 34 (refer to FIG. 1 and FIG. 2 ) for conducting imaging mass spectrometric analysis using ELDI-MS.
- the sample stage 41 While the laser beam 34 irradiates the glossy ganoderma slice to form a laser spot of 100 ⁇ 150 ⁇ m 2 thereon at an operating frequency of 10 Hz, i.e., 10 laser shots per second, the sample stage 41 is moved relative to the laser beam 34 at the speed of 0.02 cm/sec in the longitudinal direction (X), such that two subsequent laser spots formed on the glossy ganoderma slice in the longitudinal direction (X) are spaced apart from each other for 0.02 mm. The sample stage 41 was further moved in the transverse direction (Y) in consecutive increments of 1 ⁇ 6 mm upon control by the computer-controlled positioning mechanism 42 .
- the laser beam 34 scans across the glossy ganoderma slice in the longitudinal direction (X) for 60 times, each time at a different increment in the transverse direction (Y).
- each mass spectrum corresponds to an average of 10 corresponding successive laser spots that are formed on the glossy ganoderma slice and that are altogether referred to as a scanned area of the glossy ganoderma slice. Consequently, for each increment in the transverse direction (Y), 175 mass spectra were obtained. Moreover, a total of 10,500 mass spectra were obtained for the glossy ganoderma slice.
- FIGS. 6 ( a ) ⁇ ( d ) are four mass spectra of the glossy ganoderma slice obtained at various scanned areas thereof.
- a photograph of the glossy ganoderma slice identical to that shown in FIG. 4 ( a ) is illustrated on the top right hand corner of each of FIGS. 6 ( a ) ⁇ ( d ).
- An arrow is provided for each of FIGS. 6 ( a ) ⁇ ( d ) to indicate the particular scanned area of the glossy ganoderma slice that corresponds thereto.
- the intensities at these representative m/z signals in all of the mass spectra were collected.
- a molecular imaging profile is constructed for the glossy ganoderma slice at each of the representative m/z signals in the mass spectra using the computer software based on the intensities at the representative m/z signal in the mass spectra, i.e., the intensities at each of the representative m/z signals displayed by the scanned areas of the glossy ganoderma slice. Shown in FIGS.
- FIGS. 5 ( b ) ⁇ ( h ) illustrate negative images of the molecular imaging profiles shown in FIGS. 4 ( b ) ⁇ ( h ). From the molecular imaging profiles, various chemical compositions contained in the surface of the glossy ganoderma slice, and relative intensities and distributions thereof are clearly revealed.
- FIG. 7 ( a ) a slice of antrodia camphorata, a special Taiwanese fungus species that only grows on cinnamomum kanehirae, was obtained for exemplary example 2 using a razor blade, where FIG. 7 ( a ) shows a photograph of the antrodia camphorata slice.
- the antrodia camphorata slice was measured 21 mm in length, 3mm in width, and 1 mm in thickness.
- the sample stage 41 was moved relative to the laser beam 34 in the longitudinal direction (X) in the same manner as described above for exemplary example 1, such that two subsequent laser spots formed on the antrodia camphorata slice in the longitudinal direction (X) are spaced apart from each other for 0.02 mm.
- the sample stage 41 was further moved in the transverse direction (Y) in consecutive increments of 3/26 mm upon control by the computer-controlled positioning mechanism 42 .
- the laser beam 34 scans across the antrodia camphorata slice in the longitudinal direction (X) for 26 times, each time at a different increment in the transverse direction (Y).
- each mass spectrum corresponds to an average of 10 corresponding successive laser spots that are formed on the antrodia camphorata slice and that are altogether referred to as a scanned area of the antrodia camphorata slice. Consequently, for each increment in the transverse direction (Y), 105 mass spectra were obtained. Moreover, a total of 2,730 mass spectra were obtained for the antrodia camphorata slice.
- FIGS. 7 ( b ) ⁇ ( d ) are three mass spectra of the antrodia camphorata slice obtained at various scanned areas thereof.
- a corresponding arrow is provided on FIG. 7 ( a ) for each of FIGS. 7 ( b ) ⁇ ( d ) to indicate the particular scanned area on the antrodia camphorata slice that corresponds to the corresponding mass spectrum.
- the mass spectra obtained for the antrodia camphorata slice indicate two ion peak groups.
- FIGS. 8 ( b ) ⁇ ( x ) a plurality of molecular imaging profiles were constructed for the antrodia camphorata slice at each of the representative m/z signals. It is seen from FIGS. 8 ( b ) ⁇ ( e ) that volatile ions are distributed relatively evenly throughout the surface of the antrodia camphorata slice. This is because volatile odorous ions are continuously emitted from the surface of the antrodia camphorata slice, which is a tissue surface. It is seen from FIGS. 8 ( f ) ⁇ ( x ) that triterpenoid compounds concentrate more on ends of the antrodia camphorata slice (i.e., top and bottom ends of FIG.
- FIG. 10 ( a ) and FIG. 11 ( a ) two slices of angelica sinensis diels, a traditional Chinese medicine, were obtained for exemplary example 3, where FIG. 10 ( a ) shows a photograph of the angelica sinensis diels slices, and FIG. 11 ( a ) shows a negative image of FIG. 10 ( a ).
- the angelica sinensis diels slices were respectively measured 2 cm and 2 cm in length, 2 cm and 1 cm in width, and 2 mm and 2 mm in thickness.
- the sample stage 41 was moved relative to the laser beam 34 in the longitudinal direction (X) in the same manner as described above for exemplary example 1, such that two subsequent laser spots formed on each of the angelica sinensis diels slices in the longitudinal direction (X) are spaced apart from each other for 0.02 mm.
- the sample stage 41 was further moved in the transverse direction (Y) in consecutive increments of 1/15 cm for analyzing the angelica sinensis diels slices simultaneously, upon control by the computer-controlled positioning mechanism 42 .
- the laser beam 34 scans across the angelica sinensis diels slices in the longitudinal direction (X) for 30 times, each time at a different increment in the transverse direction (Y).
- each mass spectrum corresponds to an average of 10 corresponding successive laser spots that are formed on the angelica sinensis diels slices and that are altogether referred to as a scanned area of the angelica sinensis diels slices. Consequently, for each increment in the longitudinal direction (X), 200 mass spectra were obtained for the angelica sinensis diets slices. Moreover, a total of 6,000 mass spectra were obtained for the angelica sinensis diels slices.
- angelica sinensis diels has a relatively strong smell, indicating that angelica sinensis diels also contains volatile odorous chemical compositions.
- Shown in FIG. 9 is amass spectrum obtained for one of the angelica sinensis diels slices, from which two ion peak groups are found.
- FIGS. 10 ( b ) ⁇ ( n ) a plurality of molecular imaging profiles were constructed for the angelica sinensis diels slices at each of the representative m/z signals.
- FIGS. 11 ( b ) ⁇ ( n ) illustrate negative images of the molecular imaging profiles shown in FIGS. 10 ( b ) ⁇ ( n ). From FIGS. 10 ( b ) ⁇ ( c ), it is seen that the volatile odorous smaller molecular ions are distributed relatively evenly throughout the surface of the angelica sinensis diels slices, as the volatile odorous smaller molecular ions are continuously emitted from the surface of the angelica sinensis diels slices. From FIGS.
- the non-volatile ions concentrate more on outer peripheries of the angelica sinensis diels slices that correspond to an outer surface of the angelica sinensis diels from which the slices were obtained, and less near the center of the angelica sinensis diels slices that correspond to an inner portion of the angelica sinensis diels from which the slices were obtained.
- FIG. 12 ( a ) a slice of chicken brain measured 3 cm in length, 2 cm in width, and 15 ⁇ m in thickness was obtained for exemplary example 4 using the method of frozen section at ⁇ 20° C. with Shadon Cryostat (Thermo Electron, San Jose, Calif.), where FIG. 12 ( a ) shows a photograph of the chicken brain slice obtained.
- the sample stage 41 was moved relative to the laser beam 34 in the longitudinal direction (X) in the same manner as described above for exemplary example 1, such that two subsequent laser spots formed on the chicken brain slice in the longitudinal direction (X) are spaced apart from each other for 0.02 mm.
- the sample stage 41 was further moved in the transverse direction (Y) in consecutive increments of 1/30 cm upon control by the computer-controlled positioning mechanism 42 .
- the laser beam 34 scans across the chicken brain slice in the longitudinal direction (X) for 60 times, each time at a different increment in the transverse direction (Y).
- each mass spectrum corresponds to an average of 10 corresponding successive laser spots that are formed on the chicken brain slice and that are altogether referred to as a scanned area of the chicken brain slice. Consequently, for each increment in the transverse direction (Y), 150 mass spectra were obtained. Moreover, a total of 9,000 mass spectra were obtained for the chicken brain slice.
- FIGS. 12 ( b ) ⁇ ( e ) are four mass spectra of the chicken brain slice obtained at various scanned areas thereof.
- a corresponding arrow is provided on FIG. 12 ( a ) for each of FIGS. 12 ( b ) ⁇ ( e ) to indicate the scanned areas of the chicken brain slice that corresponds to the corresponding mass spectrum.
- an ion peak group with m/z values ranging approximately from 600 to 900 is found to be mainly composed of phosphatidylcholine (PC), which is a phospholipid.
- PC phosphatidylcholine
- an Optical Cutting Temperature (OCT) drug a tissue freezing medium, for embedding/imbedding the chicken brain when preparing the frozen section, is shown to surround the periphery of the chicken brain slice.
- OCT Optical Cutting Temperature
- FIGS. 13 ( a ) ⁇ ( h ) show negative images of FIGS. 13 ( b ) ⁇ ( h ), respectively.
- a chicken heart slice was obtained for exemplary example 5 using the method of frozen section at ⁇ 20° C. with Shadon Cryostat (Thermo Electron, San Jose, Calif.) With reference to FIG. 16 ( a ), the chicken heart slice was measured 25 mm in length, 18 mm in width, and 40 ⁇ m in thickness. Prior to performing imaging mass spectrometric analysis on the chicken heart slice, a saturated matrix solution, ⁇ -CHC (70% ACN, 0.1% TFA), was added evenly onto a surface thereof through 15 minutes of continued air spraying by an air-operated atomizer with 70 psi air pressure and 2.4 mL/hr solution flow rate.
- ⁇ -CHC 70% ACN, 0.1% TFA
- the sample stage 41 was moved relative to the laser beam 34 in the longitudinal direction (X) in the same manner as described above for exemplary example 1, such that two subsequent laser spots formed on the chicken heart slice in the longitudinal direction (X) are spaced apart from each other for 0.02 mm.
- the sample stage 41 was further moved in the transverse direction (Y) in consecutive increments of 0.03 mm upon control by the computer-controlled positioning mechanism 42 .
- the laser beam 34 scans across the chicken heart slice in the longitudinal direction (X) for 60 times, each time at a different increment in the transverse direction (Y).
- each mass spectrum corresponds to an average of 10 corresponding successive laser spots that are formed on the chicken heart slice and that are altogether referred to as a scanned area of the chicken heart slice. Consequently, for each increment in the transverse direction (Y), 125 mass spectra were obtained. Moreover, a total of 7,500 mass spectra were obtained for the chicken heart slice.
- FIG. 15 ( a ) is a mass spectrum of the chicken heart slice obtained at the scanned areas corresponding to fat surrounding the outer periphery of the chicken heart slice, and pointed to by an arrow.
- This mass spectrum indicates two major ion peak groups that respectively correspond to two lipid groups with molecular weight differences of 14 Da and 22 Da, respectively.
- Shown in FIGS. 16 ( c ) ⁇ ( e ) are molecular imaging profiles constructed for the chicken heart slice at the lipid representative m/z signals of the 22 Da-molecular-weight-difference group.
- Shown in FIGS. 16 ( f ) ⁇ ( h ) are molecular imaging profiles constructed for the chicken heart slice at the lipid representative m/z signals of the 14 Da-molecular-weight-difference group. Further, shown in FIGS.
- FIG. 16 ( i ) ⁇ ( m ) are molecular imaging profiles constructed for the chicken heart slice at the PC representative m/z signals.
- FIG. 17 ( a ) ⁇ ( m ) illustrate corresponding negative images of FIG. 16 ( a ) ⁇ ( m ), respectively.
- the mass spectrometric imaging method using electrospray laser assisted desorption mass spectrometry has the ability to detect molecules contained in a solid sample, such as fungus, a plant tissue, an animal tissue, etc.
- the sample Since the sample is irradiated by a laser beam, which forms a laser spot thereon, and since the sample can be maneuvered to swiftly move relative to the laser beam, the sample can be scanned by the laser beam such that irradiations of various areas thereof by the laser beam can be completed within a desirable short period of time, so as to obtain a sufficiently great number of mass spectra respectively corresponding to the scanned areas of the sample to thereby ensure that a highly accurate molecular imaging profile of the sample be obtained.
- a molecular imaging profile can be constructed to portray the spatial distribution of a particular analyte (molecule) that corresponds to the representative m/z signal. Even volatile molecules, such as odorous smaller molecules, emitted from a tissue surface can be detected, and a molecular imaging profile thereof can also be constructed.
- the mass spectrometric imaging method under ambient conditions using electrospray assisted laser desorption ionization mass spectrometry is capable of conducting imaging mass spectrometric analysis directly on solid samples.
- Molecules including non-polar molecules (e.g., triterpenoids), volatile molecules (e.g., aromatic micro-molecules), non-volatile molecules (e.g., lipids) can be detected by the present invention, and molecular imaging profiles thereof can also be constructed. Consequently, the present invention can be applied to various fields, is beneficial to basic medical science with respect to the understanding of the spatial distribution of molecules in various organs and tissues, and is especially advantageous in the diagnosis of diseases, and the discrimination between normal and abnormal tissues.
Abstract
Description
- This application is a continuation-in-part (CIP) of U.S. patent application Ser. No. 11/561,131, entitled “ELECTROSPRAY-ASSISTED LASER DESORPTION IONIZATION DEVICE, MASS SPECTROMETER, AND METHOD FOR MASS SPECTROMETRY”, filed on Nov. 17, 2006.
- 1. Field of the Invention
- The invention relates to molecular imaging, more particularly to mass spectrometric imaging under ambient conditions using electrospray-assisted laser desorption ionization mass spectrometry.
- 2. Description of the Related Art
- Imaging mass spectrometry (IMS) is widely used in the investigation of chemical or molecular distributions of solid samples, such as metals, polymers, semiconductors, and geological substances. Many attempts have been made to explore the feasibility of using imaging mass spectrometry in studying spatial distribution of proteins in various organs. However, due to the biological nature of target protein, e.g., being more labile to ionization energy and being in a state of flux, such efforts did not prove to be satisfactory.
- One of the currently-used methods of imaging mass spectrometry is the secondary ion mass spectrometry (SIMS). However, SIMS is only capable of detecting analytes such as metal ions or small organic molecules, and is unable to detect macromolecules such as peptide or proteins because the macromolecules are either spoiled during ionization or unable to be effectively desorbed from the surface of the sample.
- Another currently-used imaging method is the method of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Although MALDI-MS is capable of successfully desorbing peptide or protein molecules from a solid biological sample, and the result thereof is used to distinguish abnormal or cancerous tissues from normal tissues, several drawbacks still exist for MALDI, such as involving a tedious preparation work and requiring to be conducted in vacuum, etc.
- Yet another currently-used imaging method is the method of desorption electrospray ionization mass spectrometry (DESI-MS), which is capable of studying a variety of compounds falling within a wide range of molecular weights, and which is capable of performing direct protein mass spectrometric analysis on a freely moving tissue slice. However, there are several disadvantages involved in DESI-MS, including the difficulty in controlling the precision of striking electron-carrying spray droplets onto the tissue slice, and the inability in desorbing protein molecules from the tissue slice.
- It can be seen from the above that a variety of difficulties and inconveniences are encountered when obtaining molecular images through the methods of mass spectrometry. Since spatial analytic information of proteins in organs or tissues is extremely important in medical and biotechnological fields, there exists a need for a mass spectrometric imaging method that is capable of conducting rapid, convenient, and accurate spatial analysis on solid biological samples.
- Therefore, the object of the present invention is to provide a mass spectrometric imaging method that can be conducted under ambient conditions, and that can be used to obtain an imaging profile of a sample that has a self-sustained shape with speed and accuracy. A further object of the present invention is to provide a mass spectrometric imaging method that is capable of swiftly and un-obstructively maneuvering a sample to move relative to a desorption mechanism such that mass spectrometric results for substantially continuous areas of the sample can be obtained in a desirable short period of time.
- Another object of the present invention is to provide a mass spectrometer for implementing the mass spectrometric imaging method.
- According to one aspect of the present invention, there is provided a mass spectrometric imaging method includes the steps of: forcing sequentially generated charge-laden liquid drops to move towards a receiving unit of a mass spectrometer along a traveling path; scanning a sample with a laser beam which has an irradiation energy sufficient to cause analytes contained in the sample to be desorbed to fly along a plurality of flying paths respectively; and positioning the sample relative to the laser beam to render the plurality of flying paths intersecting the traveling path so as to permit a plurality of the analytes respectively along the plurality of flying paths to be occluded in a plurality of the charge-laden liquid drops respectively to thereby form a plurality of corresponding ionized analytes.
- Preferably, the mass spectrometric imaging method further includes the steps of obtaining a plurality of mass spectra respectively for a plurality of scanned areas of the sample through analyzing the plurality of corresponding ionized analytes which respectively correspond to the plurality of scanned areas of the sample; selecting at least one representative mass-to-charge ratio (m/z) signal which may signify a characteristic of the sample from the plurality of mass spectra; and constructing an imaging profile for the sample based on intensities at each of the at least one representative mass-to-charge ratio signal displayed by the plurality of scanned areas.
- According to another aspect of the present invention, there is provided a mass spectrometric system which is capable of obtaining an imaging profile, and which includes a mass spectrometer for analyzing ionized analytes. The mass spectrometric system includes: a receiving unit for the mass spectrometer; means for forcing sequentially generated charge-laden liquid drops to move towards the receiving unit along a traveling path; means for scanning a sample with a laser beam which has an irradiation energy sufficient to cause analytes contained in the sample to be desorbed to fly along a plurality of flying paths respectively; and means for positioning the sample relative to the laser beam to render the plurality of flying paths intersecting the traveling path so as to permit a plurality of the analytes respectively along the plurality of flying paths to be occluded in a plurality of the charge-laden liquid drops respectively to thereby form a plurality of corresponding ionized analytes
- Other features and advantages of the present invention will become apparent in the following detailed description of the preferred embodiment with reference to the accompanying drawings, of which:
-
FIG. 1 is a schematic diagram of a mass spectrometric system for implementing the preferred embodiment of a mass spectrometric imaging method under ambient conditions using electrospray-assisted laser desorption ionization mass spectrometry (ELDI-MS) according to the present invention; -
FIG. 2 is a fragmentary schematic view of the mass spectrometric system; -
FIG. 3 is a flow chart of the preferred embodiment of the mass spectrometric imaging method; -
FIG. 4 (a) illustrates a photograph of a glossy ganoderma slice obtained for conducting imaging mass spectrometric analysis in exemplary example 1; - FIGS. 4(b)˜(h) illustrate molecular imaging profiles constructed for the glossy ganoderma slice in exemplary example 1;
-
FIG. 5 (a)˜(h) illustrate negative images of FIGS. 4(a)˜(h), respectively; - FIGS. 6(a)˜(d) illustrate four mass spectra of the glossy ganoderma slice obtained at various scanned areas thereof in exemplary example 1;
-
FIG. 7 (a) is a photograph of an antrodia camphorata slice obtained for conducting imaging mass spectrometric analysis in exemplary example 2; - FIGS. 7(b)˜(d) illustrate three mass spectra of the antrodia camphorata slice obtained at various scanned areas thereof in exemplary example 2;
-
FIG. 8 (a) is another photograph of the antrodia camphorata slice; - FIGS. 8(b)˜(x) illustrate molecular imaging profiles constructed for the antrodia camphorata slice in exemplary example 2;
-
FIG. 9 illustrates a mass spectrum obtained for one of two angelica sinensis diels slices, which were obtained for conducting imaging mass spectrometric analysis in exemplary example 3; -
FIG. 10 (a) illustrates a photograph of the angelica sinensis diels slices; - FIGS. 10(b)˜(n) illustrate molecular imaging profiles constructed for the angelica sinensis diels slices in exemplary example 3;
- FIGS. 11(a)˜(n) illustrate negative images of FIGS. 10(a)˜(n), respectively;
-
FIG. 12 (a) is a diagram of a chicken brain slice obtained for conducting imaging mass spectrometric analysis in exemplary example 4; - FIGS. 12(b)˜(e) illustrate four mass spectra of the chicken brain slice obtained at various scanned areas thereof in exemplary example 4;
-
FIG. 13 (a) illustrate a diagram of the chicken brain slice with an Optical Cutting Temperature (OCT) drug that surround the periphery of the chicken brain slice; - FIGS. 13(b)˜(h) illustrate molecular imaging profiles constructed for the chicken brain slice in exemplary example 4;
- FIGS. 14(a)˜(h) illustrate negative images of FIGS. 13(a)˜(h), respectively,
-
FIG. 15 (a) illustrate a mass spectrum of a chicken heart slice, which is obtained for conducting imaging mass spectrometric analysis in exemplary example 5, at a location corresponding to fat surrounding an outer periphery of the chicken heart slice; -
FIG. 15 (b) illustrate a mass spectrum of the chicken heart slice at a location corresponding to muscle tissues at inner portions of the chicken heart slice; -
FIG. 16 (a) illustrate a photograph of the chicken heart slice; - FIGS. 16(b)˜(m) illustrate molecular imaging profiles constructed for the chicken heart slice in exemplary example 5;
- FIGS. 17(a)˜(m) illustrate negative images of FIGS. 16(a)˜(m), respectively.
- Referring to
FIG. 1 , a mass spectrometric system is used to implement the preferred embodiment of a mass spectrometric imaging method under ambient conditions using electrospray-assisted laser desorption ionization mass spectrometry (ELDI-MS) according to the present invention. The mass spectrometric system includes anelectrospray unit 2, alaser desorption unit 3, asample stage unit 4, areceiving unit 5, and an imaging processing software (not shown). - With further reference to
FIG. 2 , theelectrospray unit 2 includes areservoir 21 for accommodating a liquid electrospray medium, and anozzle 22 which is disposed downstream of thereservoir 21, and which is configured to sequentially form liquid drops of the electrospray medium thereat for traveling along a traveling path. Theelectrospray unit 2 further includes apump 23 disposed downstream of thereservoir 21 and upstream of thenozzle 22 for drawing the electrospray medium into thenozzle 22. Thenozzle 21 is spaced apart from thereceiving unit 5 in a longitudinal direction (X) so as to define the traveling path. In this embodiment, theelectrospray unit 2 further includes avoltage supplying member 24 that is disposed to establish between thenozzle 22 and the receiving unit 5 a potential difference which is of an intensity such that the sequentially formed liquid drops are laden with a plurality of charges, and such that the charge-laden liquid drops are forced to leave thenozzle 22 for heading toward thereceiving unit 5 along the traveling path. In this embodiment, the electrospray medium is an acidified methanol solution (50%). In addition, the charges laden in the liquid drops can be either univalent or multivalent. - The
laser desorption unit 3 includes a laser transmission mechanism 31 that is capable of transmitting alaser beam 34, alens 32 that is disposed to receive thelaser beam 34 from the laser transmission mechanism 31 for focusing the energy carried by thelaser beam 34, and areflector 33 that is disposed to change the path of thelaser beam 34. Thelaser desorption unit 3 is adapted to irradiate asample 6 such that, upon irradiation, a plurality of analytes, such as chemical or biochemical molecules, contained in thesample 6 are desorbed to fly along a plurality of flying paths, respectively. In this embodiment, the laser transmission mechanism 31 is a nitrogen (N2) gas laser (337 nm, 100 μJ, Q-switch). - The
sample stage unit 4 includes amovable sample stage 41 and a computer-controlledpositioning mechanism 42. Thesample stage 41 is movable relative to thelaser beam 34 such that a laser spot may be formed at a different location on thesample 6 for each laser pulse. The computer-controlledpositioning mechanism 42 is connected electrically to thesample stage 41 for controlling movement of thesample stage 41 relative to thelaser beam 34. In this embodiment, thesample stage 41 is movable in a plane along the longitudinal direction (X) and a transverse direction (Y) perpendicular to the longitudinal direction (X). It should be noted herein that thesample stage 41 can be movable in three dimensions in other embodiments of the present invention. It should be further noted that thesample stage 41 can also be made stationary, while thelaser beam 34 irradiated by the laser desorption unit 3is made movable, in other embodiments of the present invention, as long as relative movement between thesample stage 41 and thelaser beam 34 can be established. - The
sample stage 41, along with thesample 6 placed thereon, is positioned relative to thelaser beam 34 to render the plurality of flying paths of the analytes to intersect the traveling path of the charge-laden liquid drops so as to permit a plurality of the analytes respectively along the plurality of flying paths to be occluded in a plurality of the charge-laden liquid drops respectively to thereby form a plurality of corresponding ionized analytes. The ionized analytes are formed due to passing of the charges in the liquid drops onto the analytes as the charge-laden liquid drops dwindle in size when approaching the receivingunit 5 along the traveling path. - The receiving
unit 5 includes amass analyzer 51 formed with aconduit 52 that is in air communication with the environment, and adetector 53 for receiving signals generated by themass analyzer 51. Themass analyzer 51 receives the ionized analytes through theconduit 52, separates the ionized analytes according to their m/z values (mass-to-charge ratios), and generates corresponding signals for the ionized analytes. Preferably, themass analyzer 51 is selected from the group consisting of an ion trap mass analyzer, a quadrupole time-of-flight mass analyzer, a triple quadrupole mass analyzer, an ion trap time-of-flight mass analyzer, a time-of-flight/time-of-flight mass analyzer, and a Fourier transform ion cyclotron resonance (FTICR) mass analyzer. - With further reference to
FIG. 3 , the preferred embodiment of the mass spectrometric imaging method according to the present invention is performed in conjunction with the mass spectrometric system described above. - In
step 11, an object of self-sustained shape is first cut into a thin slice, which is referred to as thesample 6, by a sharp razor blade or through the method of frozen section, and thesample 6 is then placed on a stainlesssteel sample plate 7, which is disposed on thesample stage 41 of thesample stage unit 4. - In
step 12, sequentially generated charge-laden liquid drops are forced to move towards the receivingunit 5 along a traveling path. In this embodiment, the sequentially generated charge-laden liquid drops are formed by theelectrospray unit 2 at thenozzle 1 thereof, and are forced to move towards the receivingunit 5 by theelectrospray unit 2. - In
step 13, thesample 6 is irradiated with thelaser beam 34 which has an irradiation energy sufficient to cause analytes contained in thesample 6 and located at the laser spot to be desorbed to fly along a corresponding flying path. In this embodiment, thelaser beam 34 is transmitted through a fiber optic unit. - In
step 14, thesample 6 is positioned relative to thelaser beam 34 to render the flying path to intersect the traveling path so as to permit the desorbed analytes along the flying path to be occluded in the charge-laden liquid drops to thereby form a plurality of corresponding ionized analytes. - In
step 15, a mass spectrum is obtained for the scanned area of the sample 6 (i.e., the area where the laser spot is formed) through analyzing the corresponding ionized analytes which correspond to the scanned area of thesample 6. - In
step 16, thesample 6 is moved relative to thelaser beam 34 such that a different area of thesample 6 is irradiated by thelaser beam 34. -
Steps 14 to 16 are then repeated multiple times in a traceable manner. In other words, various areas of thesample 6 are irradiated by thelaser beam 34 to generate corresponding ionized analytes, and to obtain corresponding mass spectra. - In this embodiment, the
laser beam 34 is kept to irradiate along a predetermined line, and thesample stage 41 of thesample stage unit 4 is moved relative to thelaser beam 34 in incremental steps in the longitudinal direction (X) and the transverse direction (Y) by control of the computer-controlledpositioning mechanism 42, so as to position thesample 6 relative to thelaser beam 34. For instance, for every increment in the longitudinal direction (X), the computer-controlledpositioning mechanism 42 controls thesample stage 41 to move in incremental steps in the transverse direction (Y), such that various areas of thesample 6, which is placed on thesample stage 41, are sequentially irradiated by thelaser beam 34. As a result, for each scanned area of thesample 6, analytes contained in thesample 6 at the scanned area are desorbed to fly along the corresponding flying path that is rendered to intersect the traveling path of the charge-laden liquid drops so as to form the corresponding ionized analytes, and a corresponding mass spectrum is then obtained through analyzing the corresponding ionized analytes which correspond to the scanned area of thesample 6. - Consequently, by the end of these repeated steps, a plurality of mass spectra are obtained respectively for the scanned areas of the
sample 6 through analyzing the plurality of corresponding ionized analytes which respectively correspond to the scanned areas of thesample 6. - Preferably, the
laser beam 34 forms a laser spot with an area of 100 μm×150 μm on thesample 6, and the laser transmission mechanism 31 has an operating frequency of between 5 Hz to 10 Hz. With a mass spectrum corresponding to each laser spot on thesample 6, i.e., corresponding to each scanned area of thesample 6, ideally approximately eighty-two hundred to sixteen thousand mass spectra are obtained for an area of 1 cm2 on thesample 6. - In
step 17, at least one representative mass-to-charge ratio (m/z) signal which may signify a characteristic of thesample 6 is selected from the plurality of mass spectra. - In
step 18, a molecular imaging profile for thesample 6 is constructed based on intensities at each of the at least one representative m/z signal displayed by the plurality of scanned areas of thesample 6. - The present invention is described hereinafter in conjunction with a number of exemplary examples conducted to verify the mass spectrometric imaging under ambient conditions using electrospray-assisted laser desorption ionization mass spectrometry. It should be noted herein that the exemplary examples are for illustrative purposes only, and should not be taken as limitations imposed on the present invention.
- Chemicals and Equipments Used
- The exemplary examples are conducted using the following chemicals and equipments:
-
- 1. Laser Desorption Unit: The laser beams are transmitted by a pulse nitrogen laser, and has a wavelength of 337 nm, a pulse energy of 120 μJ, and an operating frequency of 10 Hz. The laser beams irradiate the
sample 6 at a 45 degree incident angle, and forms a laser focused spot size of 100×150 μm2 on the sample. - 2. Mass Analyzer (including the Detector): Ion Trap Mass Analyzer model no.
Esquire Plus 3000 plus, manufactured by Bruker Dalton company of Germany, where the mass analyzer is modified to include with a stainless steel tube with an inner diameter of 3 mm and a length of 50 mm that extends from the mass analyzer out of an entrance of the mass analyzer, and the mass spectra are obtained at a rate of one per second. - 3. Electrospray Medium: an aqueous solution containing 0.1 vol % of acetic acid and 50 vol % of methanol at a flow rate of 120 μL per hour.
- 4. Matrix: α-cyano-4-hydroxycinnamic acid (α-CHC) (70% acetonitrile (ACN), 0.1% Trifluroacetic acid (TFA)), which is a HPLC matrix manufactured by Sigma-Aldrich company of the United States.
- 5. Sample Stage Unit: the sample stage is movable at a minimum moving rate of 0.02 cm/s.
- 1. Laser Desorption Unit: The laser beams are transmitted by a pulse nitrogen laser, and has a wavelength of 337 nm, a pulse energy of 120 μJ, and an operating frequency of 10 Hz. The laser beams irradiate the
- As shown in
FIG. 4 (a) andFIG. 5 (a), a slice of glossy ganoderma, a genus of polypores, was obtained for exemplary example 1 using a razor blade, whereFIG. 4 (a) shows a photograph of the glossy ganoderma slice andFIG. 5 (a) is a negative image ofFIG. 4 (a). The glossy ganoderma slice was measured 10 mm in length, 35 mm in width, and 3 mm in thickness. The glossy ganoderma slice was placed on thesample stage 41 of the sample stage unit 4 (refer toFIG. 1 ) to be irradiated by the laser beam 34 (refer toFIG. 1 andFIG. 2 ) for conducting imaging mass spectrometric analysis using ELDI-MS. - While the
laser beam 34 irradiates the glossy ganoderma slice to form a laser spot of 100×150 μm2 thereon at an operating frequency of 10 Hz, i.e., 10 laser shots per second, thesample stage 41 is moved relative to thelaser beam 34 at the speed of 0.02 cm/sec in the longitudinal direction (X), such that two subsequent laser spots formed on the glossy ganoderma slice in the longitudinal direction (X) are spaced apart from each other for 0.02 mm. Thesample stage 41 was further moved in the transverse direction (Y) in consecutive increments of ⅙ mm upon control by the computer-controlledpositioning mechanism 42. In other words, thelaser beam 34 scans across the glossy ganoderma slice in the longitudinal direction (X) for 60 times, each time at a different increment in the transverse direction (Y). In addition, since the mass spectra were obtained at a rate of one per second, each mass spectrum corresponds to an average of 10 corresponding successive laser spots that are formed on the glossy ganoderma slice and that are altogether referred to as a scanned area of the glossy ganoderma slice. Consequently, for each increment in the transverse direction (Y), 175 mass spectra were obtained. Moreover, a total of 10,500 mass spectra were obtained for the glossy ganoderma slice. - Shown in FIGS. 6(a)˜(d) are four mass spectra of the glossy ganoderma slice obtained at various scanned areas thereof. A photograph of the glossy ganoderma slice identical to that shown in
FIG. 4 (a) is illustrated on the top right hand corner of each of FIGS. 6(a)˜(d). An arrow is provided for each of FIGS. 6(a)˜(d) to indicate the particular scanned area of the glossy ganoderma slice that corresponds thereto. - A plurality of representative m/z signals were selected from the mass spectra obtained for the glossy ganoderma slice so as to characterize the glossy ganoderma slice, and include m/z=499, m/z=513, m/z=530, m/z=553, m/z=571, m/z=1034, m/z=1047.
- With the representative m/z signals selected, the intensities at these representative m/z signals in all of the mass spectra, each of which corresponds to a different scanned area of the glossy ganoderma slice, were collected. Then, a molecular imaging profile is constructed for the glossy ganoderma slice at each of the representative m/z signals in the mass spectra using the computer software based on the intensities at the representative m/z signal in the mass spectra, i.e., the intensities at each of the representative m/z signals displayed by the scanned areas of the glossy ganoderma slice. Shown in FIGS. 4(b)˜(h) are molecular imaging profiles of the glossy ganoderma slice constructed for exemplary example 1 at the representative m/z signals thus selected (i.e., at m/z=499, m/z=513, m/z=530, m/z=553, m/z=571, m/z=1034, m/z=1047), respectively. FIGS. 5(b)˜(h) illustrate negative images of the molecular imaging profiles shown in FIGS. 4(b)˜(h).From the molecular imaging profiles, various chemical compositions contained in the surface of the glossy ganoderma slice, and relative intensities and distributions thereof are clearly revealed.
- As shown in
FIG. 7 (a), a slice of antrodia camphorata, a special Taiwanese fungus species that only grows on cinnamomum kanehirae, was obtained for exemplary example 2 using a razor blade, whereFIG. 7 (a) shows a photograph of the antrodia camphorata slice. The antrodia camphorata slice was measured 21 mm in length, 3mm in width, and 1 mm in thickness. - The
sample stage 41 was moved relative to thelaser beam 34 in the longitudinal direction (X) in the same manner as described above for exemplary example 1, such that two subsequent laser spots formed on the antrodia camphorata slice in the longitudinal direction (X) are spaced apart from each other for 0.02 mm. Thesample stage 41 was further moved in the transverse direction (Y) in consecutive increments of 3/26 mm upon control by the computer-controlledpositioning mechanism 42. In other words, thelaser beam 34 scans across the antrodia camphorata slice in the longitudinal direction (X) for 26 times, each time at a different increment in the transverse direction (Y). In addition, since the mass spectra were obtained at a rate of one per second, each mass spectrum corresponds to an average of 10 corresponding successive laser spots that are formed on the antrodia camphorata slice and that are altogether referred to as a scanned area of the antrodia camphorata slice. Consequently, for each increment in the transverse direction (Y), 105 mass spectra were obtained. Moreover, a total of 2,730 mass spectra were obtained for the antrodia camphorata slice. - Shown in FIGS. 7(b)˜(d) are three mass spectra of the antrodia camphorata slice obtained at various scanned areas thereof. A corresponding arrow is provided on
FIG. 7 (a) for each of FIGS. 7(b)˜(d) to indicate the particular scanned area on the antrodia camphorata slice that corresponds to the corresponding mass spectrum. The mass spectra obtained for the antrodia camphorata slice indicate two ion peak groups. One of the ion peak groups is composed of volatile odorous smaller molecules, and includes, for instance, m/z=107, m/z=139, m/z=167 and m/z=197. The other one of the ion peak groups is composed of triterpenoid compounds, which are active functional ingredients contained in the antrodia camphorata slice, and includes, for examples m/z=425, m/z=439, m/z=441, m/z=453, m/z=469, m/z=471 and m/z=487, etc. These m/z values were selected to be the representative m/z signals for the antrodia camphorata slice in this exemplary example. With reference to information recorded in relevant databases, the m/z=469, m/z=483, m/z=485, m/z=487, m/z=489, m/z=501, and m/z=529 ion peaks correspond to chemical compounds with chemical formulae of C31H48O3, C31H46O4, C30H44O5, C29H44O6, C31H60O4, C30H44O6, C33H52O5, respectively, and the m/z=471 ion peak corresponds to chemical compound with chemical formulae of C30H46O4, C31H50O3, C29H42O5, which respectively correspond to molecular weights of 470.68 Da, 470.73 Da and 470.64 Da. - As shown in FIGS. 8(b)˜(x), a plurality of molecular imaging profiles were constructed for the antrodia camphorata slice at each of the representative m/z signals. It is seen from FIGS. 8(b)˜(e) that volatile ions are distributed relatively evenly throughout the surface of the antrodia camphorata slice. This is because volatile odorous ions are continuously emitted from the surface of the antrodia camphorata slice, which is a tissue surface. It is seen from FIGS. 8(f)˜(x) that triterpenoid compounds concentrate more on ends of the antrodia camphorata slice (i.e., top and bottom ends of
FIG. 8 (a)) that correspond to an outer surface of the antrodia camphorata from which the slice was obtained, and less near the center of the antrodia camphorata slice that correspond to an inner portion of the antrodia camphorata from which the slice was obtained. - As shown in
FIG. 10 (a) andFIG. 11 (a), two slices of angelica sinensis diels, a traditional Chinese medicine, were obtained for exemplary example 3, whereFIG. 10 (a) shows a photograph of the angelica sinensis diels slices, andFIG. 11 (a) shows a negative image ofFIG. 10 (a). The angelica sinensis diels slices were respectively measured 2 cm and 2 cm in length, 2 cm and 1 cm in width, and 2 mm and 2 mm in thickness. - The
sample stage 41 was moved relative to thelaser beam 34 in the longitudinal direction (X) in the same manner as described above for exemplary example 1, such that two subsequent laser spots formed on each of the angelica sinensis diels slices in the longitudinal direction (X) are spaced apart from each other for 0.02 mm. Thesample stage 41 was further moved in the transverse direction (Y) in consecutive increments of 1/15 cm for analyzing the angelica sinensis diels slices simultaneously, upon control by the computer-controlledpositioning mechanism 42. In other words, thelaser beam 34 scans across the angelica sinensis diels slices in the longitudinal direction (X) for 30 times, each time at a different increment in the transverse direction (Y). In addition, since the mass spectra were obtained at a rate of one per second, each mass spectrum corresponds to an average of 10 corresponding successive laser spots that are formed on the angelica sinensis diels slices and that are altogether referred to as a scanned area of the angelica sinensis diels slices. Consequently, for each increment in the longitudinal direction (X), 200 mass spectra were obtained for the angelica sinensis diets slices. Moreover, a total of 6,000 mass spectra were obtained for the angelica sinensis diels slices. - Like antrodia camphorata, angelica sinensis diels has a relatively strong smell, indicating that angelica sinensis diels also contains volatile odorous chemical compositions. Shown in
FIG. 9 is amass spectrum obtained for one of the angelica sinensis diels slices, from which two ion peak groups are found. One of the ion peak groups is composed of volatile odorous smaller molecules, and includes, for instance, m/z=163 and m/z=191. The other one of the ion peak groups is composed of higher molecular weight chemical components, and ranges approximately from m/z=350 to m/z=500, etc. These and some additional m/z values were selected to be the representative m/z signals for the angelica sinensis diels slices in this exemplary example. - As shown in FIGS. 10(b)˜(n), a plurality of molecular imaging profiles were constructed for the angelica sinensis diels slices at each of the representative m/z signals. FIGS. 11(b)˜(n) illustrate negative images of the molecular imaging profiles shown in FIGS. 10(b)˜(n). From FIGS. 10(b)˜(c), it is seen that the volatile odorous smaller molecular ions are distributed relatively evenly throughout the surface of the angelica sinensis diels slices, as the volatile odorous smaller molecular ions are continuously emitted from the surface of the angelica sinensis diels slices. From FIGS. 10(d)˜(n), it is seen that the non-volatile ions concentrate more on outer peripheries of the angelica sinensis diels slices that correspond to an outer surface of the angelica sinensis diels from which the slices were obtained, and less near the center of the angelica sinensis diels slices that correspond to an inner portion of the angelica sinensis diels from which the slices were obtained.
- Analysis using ELDI-MS on Chicken Brain As shown in
FIG. 12 (a), a slice of chicken brain measured 3 cm in length, 2 cm in width, and 15 μm in thickness was obtained for exemplary example 4 using the method of frozen section at −20° C. with Shadon Cryostat (Thermo Electron, San Jose, Calif.), whereFIG. 12 (a) shows a photograph of the chicken brain slice obtained. Prior to performing imaging mass spectrometric analysis using the above described procedure on the chicken brain slice, a saturated matrix solution commonly used in MALDI-MS, α-CHC (70% ACN, 0.1% TFA), was added evenly onto a surface of the chicken brain slice through 3 minutes of continued air spraying by an air-operated atomizer with 70 psi air pressure and 3 mL/hr solution flow rate. Imaging mass spectrometric analysis of the present invention was conducted on the matrix-added chicken brain slice upon drying thereof. - The
sample stage 41 was moved relative to thelaser beam 34 in the longitudinal direction (X) in the same manner as described above for exemplary example 1, such that two subsequent laser spots formed on the chicken brain slice in the longitudinal direction (X) are spaced apart from each other for 0.02 mm. Thesample stage 41 was further moved in the transverse direction (Y) in consecutive increments of 1/30 cm upon control by the computer-controlledpositioning mechanism 42. In other words, thelaser beam 34 scans across the chicken brain slice in the longitudinal direction (X) for 60 times, each time at a different increment in the transverse direction (Y). In addition, since the mass spectra were obtained at a rate of one per second, each mass spectrum corresponds to an average of 10 corresponding successive laser spots that are formed on the chicken brain slice and that are altogether referred to as a scanned area of the chicken brain slice. Consequently, for each increment in the transverse direction (Y), 150 mass spectra were obtained. Moreover, a total of 9,000 mass spectra were obtained for the chicken brain slice. - Shown in FIGS. 12(b)˜(e) are four mass spectra of the chicken brain slice obtained at various scanned areas thereof. A corresponding arrow is provided on
FIG. 12 (a) for each of FIGS. 12(b)˜(e) to indicate the scanned areas of the chicken brain slice that corresponds to the corresponding mass spectrum. With reference to information recorded in relevant databases, an ion peak group with m/z values ranging approximately from 600 to 900 is found to be mainly composed of phosphatidylcholine (PC), which is a phospholipid. - As shown in
FIG. 13 (a), an Optical Cutting Temperature (OCT) drug, a tissue freezing medium, for embedding/imbedding the chicken brain when preparing the frozen section, is shown to surround the periphery of the chicken brain slice. Shown in FIGS. 13(b)˜(h) are a plurality of molecular imaging profiles constructed for the chicken brain slice at each of a plurality of representative m/z signals selected for the chicken brain slice and including m/z=332, m/z=84, m/z=735, m/z=761, m/z=790, m/z=762, and m/z=938. The OCT drug corresponds to the m/z=332 ion signal, and the molecular imaging profile obtained at m/z=332, as shown inFIG. 13 (b), clearly shows the outline of the chicken brain slice, as the OCT ions mainly concentrate around the periphery of the chicken brain slice. It can be seen from the molecular imaging profiles corresponding to m/z=84, m/z=735, m/z=761, m/z=790, m/z=762, and m/z=938, which are chemical species contained in the chicken brain slice, that these chemical species are distributed relatively evenly throughout the chicken brain slice. FIGS. 13(a)˜(h) show negative images of FIGS. 13(b)˜(h), respectively. - A chicken heart slice was obtained for exemplary example 5 using the method of frozen section at −20° C. with Shadon Cryostat (Thermo Electron, San Jose, Calif.) With reference to
FIG. 16 (a), the chicken heart slice was measured 25 mm in length, 18 mm in width, and 40 μm in thickness. Prior to performing imaging mass spectrometric analysis on the chicken heart slice, a saturated matrix solution, α-CHC (70% ACN, 0.1% TFA), was added evenly onto a surface thereof through 15 minutes of continued air spraying by an air-operated atomizer with 70 psi air pressure and 2.4 mL/hr solution flow rate. - The
sample stage 41 was moved relative to thelaser beam 34 in the longitudinal direction (X) in the same manner as described above for exemplary example 1, such that two subsequent laser spots formed on the chicken heart slice in the longitudinal direction (X) are spaced apart from each other for 0.02 mm. Thesample stage 41 was further moved in the transverse direction (Y) in consecutive increments of 0.03 mm upon control by the computer-controlledpositioning mechanism 42. In other words, thelaser beam 34 scans across the chicken heart slice in the longitudinal direction (X) for 60 times, each time at a different increment in the transverse direction (Y). In addition, since the mass spectra were obtained at a rate of one per second, each mass spectrum corresponds to an average of 10 corresponding successive laser spots that are formed on the chicken heart slice and that are altogether referred to as a scanned area of the chicken heart slice. Consequently, for each increment in the transverse direction (Y), 125 mass spectra were obtained. Moreover, a total of 7,500 mass spectra were obtained for the chicken heart slice. - Shown in
FIG. 15 (a) is a mass spectrum of the chicken heart slice obtained at the scanned areas corresponding to fat surrounding the outer periphery of the chicken heart slice, and pointed to by an arrow. This mass spectrum indicates two major ion peak groups that respectively correspond to two lipid groups with molecular weight differences of 14 Da and 22 Da, respectively. The m/z signals of the lipid ion peak groups that are selected as the representative m/z signals for the chicken heart slice include m/z=643, m/z=665, m/z=687, m/z=568, m/z=582, and m/z=596. Shown inFIG. 15 (b) is a mass spectrum of the chicken heart slice obtained at the scanned area corresponding to muscle tissues at the inner portions of the chicken heart slice, and pointed to by an arrow. This mass spectrum indicates a major ion peak group that corresponds to phosphatidylcholine (PC). The m/z signals of the PC ion peak group that are selected as the representative m/z signals for the chicken heart slice include m/z=758, m/z=760, m/z=761, m/z=768. - Shown in
FIG. 16 (b) is a molecular imaging profile constructed for the chicken heart slice at a m/z=391 background ion signal. Shown in FIGS. 16(c)˜(e) are molecular imaging profiles constructed for the chicken heart slice at the lipid representative m/z signals of the 22 Da-molecular-weight-difference group. Shown in FIGS. 16(f)˜(h) are molecular imaging profiles constructed for the chicken heart slice at the lipid representative m/z signals of the 14 Da-molecular-weight-difference group. Further, shown in FIGS. 16(i)˜(m) are molecular imaging profiles constructed for the chicken heart slice at the PC representative m/z signals.FIG. 17 (a)˜(m) illustrate corresponding negative images ofFIG. 16 (a)˜(m), respectively. - With reference to the results described hereinabove with respect to the exemplary examples, it is evident that the mass spectrometric imaging method using electrospray laser assisted desorption mass spectrometry according to the present invention has the ability to detect molecules contained in a solid sample, such as fungus, a plant tissue, an animal tissue, etc. Since the sample is irradiated by a laser beam, which forms a laser spot thereon, and since the sample can be maneuvered to swiftly move relative to the laser beam, the sample can be scanned by the laser beam such that irradiations of various areas thereof by the laser beam can be completed within a desirable short period of time, so as to obtain a sufficiently great number of mass spectra respectively corresponding to the scanned areas of the sample to thereby ensure that a highly accurate molecular imaging profile of the sample be obtained.
- In addition, by integrating the results from all of the mass spectra, spatial distribution profiles of the molecules contained in the sample can be generated. More particularly, based on the intensities at a selected representative mass-to-charge ratio (m/z) signal displayed by a plurality of scanned areas of the sample, a molecular imaging profile can be constructed to portray the spatial distribution of a particular analyte (molecule) that corresponds to the representative m/z signal. Even volatile molecules, such as odorous smaller molecules, emitted from a tissue surface can be detected, and a molecular imaging profile thereof can also be constructed.
- In sum, the mass spectrometric imaging method under ambient conditions using electrospray assisted laser desorption ionization mass spectrometry according to the present invention is capable of conducting imaging mass spectrometric analysis directly on solid samples. Molecules including non-polar molecules (e.g., triterpenoids), volatile molecules (e.g., aromatic micro-molecules), non-volatile molecules (e.g., lipids) can be detected by the present invention, and molecular imaging profiles thereof can also be constructed. Consequently, the present invention can be applied to various fields, is beneficial to basic medical science with respect to the understanding of the spatial distribution of molecules in various organs and tissues, and is especially advantageous in the diagnosis of diseases, and the discrimination between normal and abnormal tissues.
- While the present invention has been described in connection with what is considered the most practical and preferred embodiment, it is understood that this invention is not limited to the disclosed embodiment but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.
Claims (8)
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JP2007253799A JP2009042206A (en) | 2007-07-19 | 2007-09-28 | Mass spectrometric imaging method and mass spectrometer system |
RU2007140231/28A RU2007140231A (en) | 2007-07-19 | 2007-10-30 | METHOD FOR FORMING MASS-SPECTROMETRIC IMAGE UNDER ENVIRONMENT USING MASS-SPECTROMETRY USING LASER DESORPTION IONIZATION BY ELECTRON SPRAYING |
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US11/561,131 US7696475B2 (en) | 2006-01-27 | 2006-11-17 | Electrospray-assisted laser desorption ionization device, mass spectrometer, and method for mass spectrometry |
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